HIV-1 gag matrix (gag MA), the N-terminal product of gag Pr55 polyprotein, is a multifunctional structural protein which is involved in late stages of the viral lifecycle. Gag MA also appears to function in viral infectivity. We have previously demonstrated that, following virus infection, gag MA associates with viral reverse transcription complexes, is phosphorylated and localizes to the nucleus. We have proposed a model-implicating gag MA in nuclear targeting of the viral reverse transcription (RT) complex, a function which is important for the ability of HIV-1 to access the intact nucleus of nondividing cells such as macrophages. However, gag MA does not appear to satisfy the biochemical criterion for a nuclear protein in that gag MA localizes to the cytoplasm when expressed in mammalian cells in the absence of other viral proteins. In addition, a model invoking gag MA as an infectivity factor creates a paradox in that gag MA, by virtue of its myristoylation moiety, targets gag precursors to the plasma membrane, the site of virus assembly. Thus, these opposing targeting functions must somehow be coordinated at distinct stages of the virus lifecycle. In the previous funding period, we have obtained experimental evidence to support the notion that gag MA plays a critical role in viral infectivity. Gag MA exhibits nucleocytoplasmic shuttling activity which is essential in the viral lifecycle. Our data suggests the presence of a nuclear export signal (NES) in gag MA which, when inactivated, results in accumulation of gag precursors and of genomic viral RNA in the host cell nucleus. We propose that the gag MA NES counteracts nuclear targeting of gag MA in the virus-producing cell to ensure cytoplasmic availability of virion components during virus assembly. In the next funding period, we propose to more fully characterize how nucleocytoplasmic shuttling activity of gag MA functions in the virus lifecycle. Specifically, we propose to:
Specific Aim 1 : Identify effector domains in gag MA that govern its nucleocytoplasmic shuttling.
Specific Aim 2 : Characterize cellular nuclear import and export factors that mediate nucleocytoplasmic shuttling of gag MA.
Specific Aim 3 : Characterize the mechanism by which gag MA nuclear export and nuclear import activity is coordinated during early and late phases of the virus lifecycle.
Specific Aim 4 : Examine the role of nucleocytoplasmic shuttling activity in the virus lifecycle both in vitro and in vivo. It is expected that these studies will more fully define how gag MA participates in early and late phases of the viral lifecycle.
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